Alginate is an unbranched polysaccharide containing two epimers of uronic acid [1]. Mannuronic acid (M) and its C-5-epimer guluronic acid (G) are linked by α and β (1→4) glycosidic bonds [2]. The composition of an alginate can be calculated by nearest neighbour diad frequency using 13C nuclear magnetic resonance (NMR) spectroscopy [3]. Brown seaweed is a major commercial source of alginate, where it occurs as a structural component of the cell wall. The arrangements of the acid residues are of great importance as they confer specific characteristics. Higher levels of M residues give an increase in flexibility of that section of seaweed. For example, the highest level of M residues of Laminaria hyperboria is found in the leaf tissue whereas the stipe is much lower in M residues, with the outer cortex even lower [4]. Alginates are not only exploited by algae, but also by bacteria. In the soil bacteria, Azotobacter vinlandii, alginates are the major constituents of the vegetative capsule and of the rigid and desiccation-resistant walls of the metabolically dormant cysts [5]. The alginates produced by many bacteria, for example Pseudomonas spp. appear to have multiple roles in environmental protection whereas both bacterial and seaweed alginates can stimulate the human immune system [5].
Enzymes that possess the ability to epimerise M residues to G residues have been isolated and purified from bacterial strains that utilise alginates. A family of seven epimerase enzymes (AlgE1-7) have been purified from A. vinlandii and their activity described. Each enzyme is likely to produce a distinct monomer distributions pattern, for example AlgE1 is the only double epimerasic enzyme creating two consecutive G residues whereas the other epimerase enzymes can only affect one residue at a time. Bacterial alginates can be created as homopolymeric M residue polymers and can then be processed by the enzymes to their desired characteristics. Epimerase negative stains of Pseudomonas fluorescens have been developed which allows alginates of purely M residues to be harvested when the bacteria are grown on media containing a high concentration of D-fructose-6-phosphate [6].
A varying range of lipases are produced within the human body as well as by bacteria, all of which are responsible for the catalysis of hydrolysis of ester bonds in triacylglycerols. With the exception of pancreatic lipase, all lipases are single domain enzymes. Pancreatic lipase requires another protein (colipase) for activity in the presence of bile salts or detergents [7]. Colipase (11000 Da) is involved with the activity of the enzyme, prevention of denaturation at the water-lipid interface and reverses the inhibitory effect of bile salts at the same interface. There are two conformations of pancreatic lipase, the open (active) form and the closed (inactive) form [8]. The conformation is changed via the movement of two loops of amino acids uncovering the hydrophobic active site. The binding of the colipase does not initiate activity of the enzyme nor does it initiate the movement of the loops but, when the colipase is bound at the lipid-water interface, the loops make multiple contacts with the colipase when in the open conformation [8]. There is a common fold throughout all the lipases termed the α/β hydrolase fold due to the orientation of the α-helices and the arrangement of the β-strands. The active site of the pancreatic lipase is composed of a catalytic serine-histadine-aspartate triad and this triad is well conserved throughout the lipase family [8]. The lipid substrate is likely to enter the active site in a ‘tuning fork’ orientation [9] with one acyl chain (one prong) in the active site and the second acyl chain (second prong) running along the outside of the lipase molecule in a groove created by two phenylalanine residues [10].
Some pharmacological obesity treatments, e.g. Orlistat (Trade Mark), function through specific, irreversible inhibition of gastrointestinal lipases, of which pancreatic lipase is the most biologically active and important in healthy humans [11]. A number of adverse effects are commonly reported for Orlistat, including steatorrhoea, bloating, oily spotting, faecal urgency and faecal incontinence that can affect up to 40% of patients [12]. This leads to high attrition rates and tolerability problems. A product which retains the level of lipase inhibition but reduces or eliminates the adverse effects of current treatment would be of considerable benefit to patients.